Pathological mechanisms of hepatic ischemia-reperfusion injury and stem cell-based therapeutic strategies: Mechanistic insights and translational perspectives.

Hepatic ischemia reperfusion injury (IRI) remains a major obstacle in liver resection and transplantation surgery, especially in diseased organs. Human mesenchymal stromal cells (MSCs) are reported to acutely alleviate hepatic IRI in mice by releasing bioactive membrane-enclosed extracellular vesicles (EVs), but the long-term effects of MSC-derived EV on hepatic IRI are unknown. Given the considerable differentiation capacity of fibroblasts (FBs) during wound healing and their morphological similarities with MSC, the present study aimed to investigate the potential of these two cell types and their cell-derived EV in attenuating liver damage after IRI. EVs were isolated and purified from the supernatant of MSC and FB cultures and, subsequently, characterized by electron microscopy, nanoparticle tracking analysis, and western blot. Liver injury and organ regeneration in a murine in vivo model of IRI were assessed by serum transaminase levels, histopathology, and immunohistochemistry. Changes in expression of inflammation-associated genes within liver tissue were evaluated by reverse transcriptase quantitative polymerase chain reaction. MSC, MSC-derived EV, FB, and FB-derived EV were systemically administered before hepatic IRI. We found that MSC and MSC-derived EV decreased serum transaminase levels, reduced hepatic necrosis, increased the amount of Ki67-positive hepatocytes, and repressed the transcription of inflammation-associated genes. Although they had no impact on organ damage, FB and FB-derived EV showed some regenerative potential in the late phase of hepatic IRI. Compared to FB, MSC and their derived EV had a stronger potential to attenuate liver damage and improve organ regeneration after hepatic IRI. These results suggest that the key therapeutic factors are located within EV.

The therapeutic potential of mesenchymal stem cell (MSC) extracellular vesicles (EV) is currently under investigation in many pathological contexts. Both adult and perinatal MSC are being considered as sources of EV. Herein, we address antigen expression of cord blood and bone marrow MSC and released EV to define an identity and quality parameter of MSC EV as a medicinal product in the context of clinical applications. The research focuses on EV-shuttled neural/glial antigen 2 (NG2), which has previously been detected as a promising surface marker to distinguish perinatal versus adult MSC. Indeed, NG2 was significantly more abundant in cord blood than bone marrow MSC and MSC EV. Ultracentrifuge-isolated EV were then challenged for their pro-angiogenic properties on an xCELLigence system as quality control. NG2+ cord blood MSC EV, but not bone marrow MSC EV, promote bFGF and PDGF-AA proliferative effect on endothelial cells. Likewise, they successfully rescue angiostatin-induced endothelial cell growth arrest. In both cases, the effects are NG2-dependent. These results point at NG2 as an identity and quality parameter for cord blood MSC EV, paving the way for their clinical translation.

SIRT1 alleviates hepatic ischemia-reperfusion injury via the miR-182-mediated XBP1/NLRP3 pathway

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

Mesenchymal Stem Cells Over-expressing Hepatocyte Growth Factor Improve Small-for-size Liver Grafts Regeneration

Mesenchymal stem cells (MSCs) have been shown to mitigate vascular permeability in hemorrhagic shock (HS) and trauma-induced brain and lung injury. Mechanistically, paracrine factors secreted from MSCs have been identified that can recapitulate many of the potent biologic effects of MSCs in animal models of disease. Interestingly, MSC-derived extracellular vesicles (EVs), contain many of these key soluble factors, and have therapeutic potential independent of the parent cells. In this study we sought to determine whether MSC-derived EVs (MSC EVs) could recapitulate the beneficial therapeutic effects of MSCs on lung vascular permeability induced by HS in mice. Mesenchymal stem cell EVs were isolated from human bone marrow-derived MSCs by ultracentrifugation. A mouse model of fixed pressure HS was used to study the effects of shock, shock + MSCs and shock + MSC EVs on lung vascular endothelial permeability. Mice were administered MSCs, MSC EVs, or saline IV. Lung tissue was harvested and assayed for permeability, RhoA/Rac1 activation, and for differential phosphoprotein expression. In vitro, human lung microvascular cells junctional integrity was evaluated by immunocytochemistry and endothelial cell impedance assays. Hemorrhagic shock-induced lung vascular permeability was significantly decreased by both MSC and MSC EV infusion. Phosphoprotein profiling of lung tissue revealed differential activation of proteins and pathways related to cytoskeletal rearrangement and regulation of vascular permeability by MSCs and MSC EVs. Lung tissue from treatment groups demonstrated decreased activation of the cytoskeletal GTPase RhoA. In vitro, human lung microvascular cells, MSC CM but not MSC-EVs prevented thrombin-induced endothelial cell permeability as measured by electrical cell-substrate impedance sensing system and immunocytochemistry of VE-cadherin and actin. Mesenchymal stem cells and MSC EVs modulate cytoskeletal signaling and attenuate lung vascular permeability after HS. Mesenchymal stem cell EVs may potentially be used as a novel "stem cell free" therapeutic to treat HS-induced lung injury.

Functionally engineered extracellular vesicles improve bone regeneration

Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from the full potential of hMSC. In this current review, we present some of the most important and recent advances in understanding of the biology of hMSC and their current and potential use in therapy.

3D Encapsulation and tethering of functionally engineered extracellular vesicles to hydrogels

BackgroundPatients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity in vitro and in vivo in a mouse model of HS/T. This study addresses the systemic effects of HS/T on multiorgan EOT.MethodsIn vitro, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (H2O2) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. In vivo, a mouse model of HS/T was used to evaluate the effects of lactated Ringer’s (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers.ResultsMSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer in vitro, whereas only MSC CM was protective of the Caco-2 epithelial monolayer. In vivo, both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice.ConclusionsIn conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies.

BACKGROUNDPatients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity in vitro and in vivo in a mouse model of HS/T. This study addresses systemic effects of HS/T on multiorgan EOT in HS/T model.METHODSIn vitro, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (H2O2) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. In vivo, a mouse model of HS/T was used to evaluate the effects of lactated Ringer’s (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers.RESULTSMSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer in vitro, whereas only MSC CM was protective of the Caco-2 epithelial monolayer. In vivo, both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice.CONCLUSIONSIn conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies.

Mesenchymal stem cell derived extracellular vesicles (MSC EVs) possess excellent immunomodulatory and therapeutic properties. While beneficial, from a translational perspective, extracellular vesicles with consistent functionality and target specificity are required to achieve the goals of precision medicine and tissue engineering. Prior research has identified that the miRNA composition of mesenchymal stem cell derived extracellular vesicles contributes significantly towards extracellular vesicles functionality. In this study, we hypothesized that mesenchymal stem cell derived extracellular vesicle functionality can be rendered pathway-specific using a miRNA-based extracellular vesicles engineering approach. To test this hypothesis, we utilized bone repair as a model system and the BMP2 signaling cascade as the targeted pathway. We engineered mesenchymal stem cell extracellular vesicles to possess increased levels of miR-424, a potentiator of the BMP2 signaling cascade. We evaluated the physical and functional characteristics of these extracellular vesicles and their enhanced ability to trigger the osteogenic differentiation of naïve mesenchymal stem cell in vitro and facilitate bone repair in vivo. Results indicated that the engineered extracellular vesicles retained their extracellular vesicles characteristics and endocytic functionality and demonstrated enhanced osteoinductive function by activating SMAD1/5/8 phosphorylation and mesenchymal stem cell differentiation in vitro and enhanced bone repair in vivo. Furthermore, the inherent immunomodulatory properties of the mesenchymal stem cell derived extracellular vesicles remained unaltered. These results serve as a proof-of-concept for miRNA-based extracellular vesicles engineering approaches for regenerative medicine applications.

SummaryChronic liver diseases, including cirrhosis and liver failure, remain formidable challenges due to their complex progression and limited therapeutic options. Mesenchymal stem cell (MSC) therapy has emerged as a game-changing approach, leveraging its potent immunomodulatory, anti-fibrotic, and regenerative capabilities, along with the ability to transdifferentiate into hepatocytes. This review delves into the latest advances in MSC-based treatments for chronic and end-stage liver diseases, as highlighted in current clinical trials. MSCs derived from bone marrow and umbilical cord have shown remarkable promise in reversing liver damage, improving liver function, and providing hope for patients who do not respond to conventional therapies. When administered through hepatic, portal, or peripheral veins, MSCs have significantly improved liver histology, reduced fibrosis, and restored functional capacity. Furthermore, MSC-derived materials, such as extracellular vesicles and exosomes, are emerging as cutting-edge tools for treating liver failure and mitigating post-transplant complications. While autologous MSC-derived hepatocytes hold promise for non-fatal cirrhosis, allogeneic MSCs are being applied in more severe conditions, including liver failure and transplantation cases. Despite these promising early outcomes, larger trials and long-term studies are essential to fully harness MSCs as a transformative, off-the-shelf alternative to liver transplantation, heralding a new era in regenerative liver therapies.

Introduction: Mesenchymal Stem Cell (MSC)-derived Extracellular Vesicles (EVs) are an emerging regenerative therapy for treatment of ischemic cardiomyopathy. In this study, we determine the efficacy of MSC-EV therapy in a shear-thinning hydrogel (STG) delivered via intramyocardial injection to the border zone of rat hearts following myocardial infarction (MI). Hypothesis: MSC EV-loaded STGs will preserve hemodynamic function and minimize ventricular scar formation in a rat model of acute MI. Methods: EVs were isolated from MSCs by PEG precipitation, and the EV proteome was characterized by MaxQuant 1.5.1.2 and Metacore analysis software. Varying concentrations of EVs were administered to rats following induction of acute MI by ligation of the left anterior descending artery. EVs were delivered in either sterile phosphate buffered saline (PBS) or STG for sustained EV release. Chronic injury was assessed at 4 weeks post-MI through transthoracic echocardiography, intraventricular pressure-volume loop displacement, and histology. Results: MSC EV proteomic analysis highlighted upregulation of VEGF angiogenic cascades and ILK-mediated proliferative pathways. The mean left ventricle ejection fraction (LVEF) for each treatment group at four weeks is displayed in Table 1. A 20 ug dose of MSC EVs in STG improved LVEF by 19.12% (p<0.05) compared to PBS, with a 90 ug dose of MSC EVs in STG increasing LVEF by 23.56% (p<0.01) (Fig. 1). Conclusions: Intramyocardial injection of MSC EVs in STG post-MI showed concentration dependent improvements in hemodynamics. These findings show that high doses of MSC EV in STG may have potential as a therapy for ischemic cardiomyopathy. Proteomic analysis revealed that angiogenesis, recruitment of cardiac progenitor cells, and immune modulation may be mechanistic drivers of MSC-EV therapy.

Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have beneficial effects in preclinical bronchopulmonary dysplasia and pulmonary hypertension (BPD-PH) models. The optimal source, dosing, route, and duration of effects are however unknown. The objectives of this study were to (a) compare the efficacy of GMP-grade EVs obtained from Wharton’s Jelly MSCs (WJ-MSCs) and bone marrow (BM-MSCs), (b) determine the optimal dosing and route of administration, (c) evaluate its long-term effects, and (d) determine how MSC EVs alter the lung transcriptome. Newborn rats exposed to normoxia or hyperoxia (85% O2) from postnatal day (P)1-P14 were given (a) intra-tracheal (IT) BM or WJ-MSC EVs or placebo, (b) varying doses of IT WJ-MSC EVs, or (c) IT or intravenous (IV) WJ-MSC EVs on P3. Rats were evaluated at P14 or 3 months. Early administration of IT BM-MSC or WJ-MSC EVs had similar beneficial effects on lung structure and PH in hyperoxia-exposed rats. WJ-MSC EVs however had superior effects on cardiac remodeling. Low, medium, and high dose WJ-MSC EVs had similar cardiopulmonary regenerative effects. IT and IV WJ-MSC EVs similarly improved vascular density and reduced PH in hyperoxic rats. Gene-set enrichment analysis of transcripts differentially expressed in WJ-MSC EV-treated rats showed that induced transcripts were associated with angiogenesis. Long-term studies demonstrated that a single early MSC EV dose has pulmonary vascular protective effects 3 months after administration. Together, our findings have significant translational implications as it provides critical insight into the optimal source, dosing, route, mechanisms of action, and duration of effects of MSC-EVs for BPD-PH.